Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export

RNA undergoing nuclear export first encounters the basket of the nuclear po re. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, b ut their molecular partners within the pore are largely unknown. Because th e mechanism of RNA export will be in question as long as significant verteb rate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenop us egg extracts, which contain abundant disassembled nuclear pores. Strikin gly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleopor ins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced . Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleopor ins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg ext racts and in assembled pores, now termed the Nup160 complex. Sec13 is promi nent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nu p160 complex. We have mapped the sites that are required for binding the Nu p160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find t hat specific Nup160 and Nup133 fragments block poly[A](+) RNA export, but n ot protein import or export. These results demonstrate that two novel verte brate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup 153, but themselves play a role in mRNA export.